The cells of our bodies accumulate storage lipids in the organelles called lipid droplets (LDs). LDs play an important role in lipid metabolism. Their abundance is controlled by lipolysis via cytosolic lipases and by lipophagy, the autophagic trafficking pathway that delivers LDs to lysosomal/vacuolar lipases. The lack of lipolysis or lipophagy creates an excess of LDs causing obesity and obesity-related disorders. The recent discovery of lipophagy opened a new opportunity for treatment of obesity, but our knowledge of this pathway is still very limited. During the K01 award project, we isolated a yeast mutant with highly elevated basal lipophagy. Therefore, the first goal of this project is to identify and validte the gene responsible for this phenotype. If this gene is conserved in mammals, we will also validate the role of this gene in the regulation of mammalian lipophagy. Our second goal is to identify the morphological intermediates and proteins essential for lipophagy. This will provide us with essential framework for a subsequent R01 grant application and in-depth studies on the role of the identified regulator in lipophagy. Reaching these goals might also provide a new promising target for prevention and treatment of obesity, since pharmaceutical inhibition of the lipophagy suppressor will increase the rates of lipophagy and decrease the volume of LDs. Our project has two specific aims. Aim 1 is dedicated to identification and validation of the negative regulator of lipophagy. It is a logical extension of the K01's Aim 3 where we developed the Pichia pastoris yeast as a simple lipophagy model and isolated the nrl1 mutant affected in the Negative Regulation of Lipophagy. We will sequence the genome of nrl1 cells and identify the gene(s) affected by mutation(s). Then, we will narrow them down to the gene of the lipophagy suppressor by gene deletion analysis. To validate the identified gene as the gene of the lipophagy suppressor, we will perform the gene overexpression studies. If the identified gene is conserved in mammals, we will also validate its role in lipophagy through gene knockdown and overexpression studies using rat hepatocytes and established lipophagy assays. Aim 2 will identify the morphological intermediates and molecular architecture of lipophagy. In the K01's Aim 3, we found that lipophagy in P. pastoris depends on the autophagy-related proteins, Atg5 and Atg8, and on the vacuolar sequestering membranes. Here, we will examine if engulfment of LDs by the vacuolar sequestering membranes involves formation of a bridging autophagic membrane, the phagophore. We will also define the lipophagy requirements of all known Atg-proteins and identify the stage of the pathway blocked in each atg-mutant. Finally, we will build a molecular model of lipophagy with potential points of positive and negative regulation, which will be further explored in the R01 grant application.